The overall goal of this project is to characterize the genome sequences associated with diverse isolates of Yersinia pestis, Burkholderia pseudomallei, and Francisella tularensis at a level of comprehensiveness not yet achieved for any bacterial species. Conventional whole-genome sequencing will be carried out on at least one additional strain of each species to complement data from existing projects. However, the main focus will be on targeted resequencing of genome segments from large numbers of strains. The proposed technology relies on fosmid clones, a recombinant DNA system suitable for cloning genome segments approximately 40 kbp in size. Randomly picked fosmids will be characterized by end-sequencing and restriction-digest fingerprinting; a sufficient number of clones will be analyzed to provide deep (approx. 10X) whole-genome coverage of each bacterial strain analyzed. Alignment of fosmid end sequences and digests with available reference sequences will allow detection of genomic regions in new strains that are highly diverged or rearranged relative to the reference sequences. Fosmids from these regions will be completely sequenced. The data will also be analyzed to obtain a comprehensive view of the patterns of genetic variation across the whole genome of each strain analyzed. We expect the project to exemplify a new standard for thorough analysis of the genomes of major bacterial species. U!timately, the goal will be to create a sufficiently large and diverse database of reference sequences--some of whole genomes others of highly variable genome segments--that most genes and gene arrangements encountered in newly isolated strains will be represented in the database.